Metabolic reprogramming, the shift from oxidative to glycolytic metabolism, has been increasingly considered as a core hallmark of cancer. Our recent in vitro study suggests that metabolic reprogramming may be the underlying mechanism through which oncogenic Ras-induced mitochondrial dysfunction drives tumor initiation and progression. Examining this innovative and important hypothesis holds great promise to advance our understanding of the metabolic signaling in cancer and inspire new strategies for early cancer detection and targeted therapy. However, it has been impeded by the lack of a technology capable of dynamically imaging the metabolic shift at the microscopic level in vivo. The proposed research aims to meet this stringent demand by developing a reflection-mode dual-modal microscopy platform. Integrating two cutting-edge techniques- photoacoustic microscopy (PAM) and multiphoton fluorescence lifetime imaging microscopy (MPFLIM)-in a radically new way, this platform will enable concurrent imaging of two gold-standard metabolic indices: the metabolic rate of oxygen (MRO2) and optical redox ratio. Co-evolution of the two indices will quantitatively and dynamically delineate metabolic reprogramming during cancer development. To this end, we have organized this project around three specific aims. First, we will develop an optical-acoustic objective by integrating a high- frequency ultrasonic transducer and a reflective microscope objective. This novel design will enable the natural integration of optical excitation (for both PAM and MPFLIM) and acoustic detection (for PAM only) in reflection mode. Then, we will implement the dual-modal microscopy platform based on our existing MPFLIM system. Concurrent detection of optically-absorbing blood hemoglobin and autofluorescent metabolic coenzymes (i.e., NADH and FAD) through properly designed dual-modal scan and data acquisition will enable, for the first time, in vivo high-resolution imaging of MRO2 and optical redox ratio at the same spatiotemporal scale. Ultimately, validating the performance of the platform in a mouse tumor model will prepare us for follow-up mechanistic studies of the role of Ras-induced mitochondrial dysfunction in metabolic reprogramming and tumorigenesis. Successful completion of this developmental grant will lead to a technical breakthrough in the field of metabolic imaging and allow us to tackle important questions, including whether genetic inhibition of mitochondrial fission can reverse metabolism reprogramming and which genetic factors contribute to the metabolic phenotypes in cancer. These studies not only will give us a clearer understanding of cancer metabolic signaling but also may identify new therapeutic targets for the treatment of Ras-driven malignancies. The potential impact of the dual- modal microscopy could be very broad, because metabolic reprogramming contributes to the development of numerous diseases besides cancers, including but not limited to Alzheimer disease, obesity, and diabetes. The robust performance and ease of implementation, enabled by the novel optical-acoustic objective, will pave the way for disseminating this promising technique throughout the research community.